Gaucher's disease is an autosomal recessive lysosomal storage disorder characterized by a deficiency in the lysosomal enzyme, glucocerebrosidase (GCR). GCR hydrolyzes the glycolipid glucocerebroside that is formed after degradation of glucosphingolipids in the membranes of white blood cells and senescent red blood cells. In patients with Gaucher's disease, deficiency in this enzyme causes the glucocerebroside to accumulate in large quantities in the lysosomes of phagocytic cells located in the liver, spleen and bone marrow. Accumulation of these molecules causes a range of clinical manifestations including splenomegaly, hepatomegaly, skeletal disorder, thrombocytopenia and anemia.
Former treatments for patients suffering from this disease include administration of analgesics for relief of bone pain, blood and platelet transfusions and, in several cases, splenectomy. Joint replacements were sometimes necessary for patients who experienced bone erosion. In 1966, Brady published an article in The New England Journal of Medicine (vol. 275, p. 312) proposing enzyme replacement therapy with GCR as a treatment for Gaucher's disease. The current treatment of patients with Gaucher's disease relies on the administration of a carbohydrate remodelled GCR derived from the placenta, known as p-GCR. The carbohydrate remodelling converts the native molecule into a molecule that will bind to mannose receptors on phagocytic cells. While enzyme replacement therapy using remodelled p-GCR has been shown to be effective in treating patients, it is an expensive form of therapy and places a heavy economic burden on the health care system. The high cost of p-GCR results from the scarcity of the human placental tissue from which it is derived, a complex purification protocol, and the relatively large amounts of the therapeutic required for existing treatments. There is an urgent need to reduce the cost of GCR so that this life saving therapy can be provided to all who require it more affordably.
Unmodified glucocerebrosidase derived from natural sources is a glycoprotein with four carbohydrate chains. This protein does not target the phagocytic cells in the body and is therefore of limited therapeutic value. In developing the current therapy for Gaucher's disease, the terminal sugars on the carbohydrate chains of glucocerebrosidase are sequentially removed by treatment with three different glycosidases. This glycosidase treatment results in a glycoprotein whose terminal sugars consist of mannose residues. Since phagocytes have mannose receptors that recognize glycoproteins and glycopeptides with oligosaccharide chains that terminate in mannose residues, the carbohydrate remodeling of glucocerebrosidase has improved the targeting of the enzyme to these cells (Furbish et al., Biochem. Biophys. Acta 673:425, 1981)
This approach for improving the efficacy of glucocerebrosidase by remodelling the carbohydrate chains does not exclusively target glucocerebrosidase to phagocytes, such as Kupffer cells, in preference to other cells since other cells, such as sinusoid endothelial cells, have mannose receptors. These cells very likely compete with phagocytes for uptake of p-GCR by this receptor mediated pathway. In addition, there is considerable uptake of p-GCR by hepatocytes by a non-mannose receptor pathway(s). Uptake by non-phagocytic cells does not provide therapeutic value. As a result, p-GCR is administered in high doses, and the cost is considerable.
Until this time, the focus for achieving reduced cost for enzyme replacement therapy has been to obtain GCR from alternative sources to the placenta, the sources being either natural or synthetic (using recombinant DNA technology) (Sorge et al., P.N.A.S., 82:7289, 1985, and Tsuji et al., J. Biol. Chem., 261:50, 1986). However, at least as important is the ability to direct the enzyme more effectively to target cells using new methods for administration of the enzyme and novel compositions that favor improved pharmacokinetics. This approach has not previously been systematically investigated in vivo.
A means of determining how much modified GCR is delivered and taken up by target cells and non-target cells in the liver would be helpful in evaluating further modifications to the composition, formulation and administration of GCR and determining the cost benefit of these improvements for the patients. This involves a method for separating Kupffer cells from other non-parenchymal cells isolated from the liver. However, the isolation of specific cell types from an experimental animal can be technically complex using conventional techniques of separation that rely on size separation when different cell types are similar sized as found with Kupffer cells and endothelial cells in the liver. In these circumstances, it would be desirable to develop accurate and relatively simple methods to recover target cells such as Kupffer cells in order to measure uptake of exogenous GCR, and subsequently to determine the half life of the GCR at the site at which it acts in vivo.
For the foregoing reasons, new methods for treating patients with GCR that incorporate an understanding of the pharmacokinetics of the drug are required to provide treatments that are more cost effective than existing treatments for Gaucher's disease so as to provide life saving therapy to all who need it.